重组荞麦胰蛋白酶抑制剂对HL-60细胞的促凋亡作用

为了研究基因重组荞麦胰蛋白酶抑制剂诱导HL-60细胞凋亡的作用并探讨其抗癌作用机理,用不同浓度的重组荞麦胰蛋白酶抑制剂体外作用于HL-60细胞,采用MTT比色法检测其对HL-60细胞的抑制率,应用光学显微镜观察细胞核的形态学变化,用DNA凝胶电泳法检测HL-60细胞的凋亡,用流式细胞术检测该抑制剂作用于HL-60细胞后引起的凋亡现象。结果表明：重组荞麦胰蛋白酶抑制剂对HL-60细胞的生长具有明显的抑制作用,且随着蛋白质浓度的增加对HL-60细胞生长抑制愈加明显,而对正常人外周血单个核细胞（PBMNC）无作用;荧光显微镜下观察发现经该蛋白作用后HL-60细胞核的形态呈现凋亡特征;流式细胞术分析表明,HL-60细胞经100μg/ml重组荞麦胰蛋白酶抑制剂作用后,凋亡率达到52％;琼脂糖凝胶电泳显示细胞DNA呈梯状降解。结论：重组荞麦胰蛋白酶抑制剂在体外能够明显抑制HL-60细胞的增长,并诱导细胞凋亡,这为重组荞麦胰蛋白酶抑制剂应用于急性髓细胞性白血病的治疗提供依据,也为重组基因工程植物蛋白药物的临床应用提供新思路。

Apoptosis of HL-60 Cells Induced by Recombinant Common Buckwheat Trypsin Inhibitor

The study was purposed to investigate the apoptosis of HL-60 cells induced by recombinant common buckwheat trypsin inhibitor (rBTI) and its mechanism. The inhibition rate of rBTI on HL-60 cells was detected by MTT (3-4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide); the morphology of HL-60 nuclei was observed by fluorescence microscopy; the apoptosis cells of HL-60 detected by agarose gel electrophoresis and the changes of apoptosis rate was assayed by flow cytometry (FCM), when the HL-60 cells were treated with different concentration of rBTI for 24 hours. The results showed that the growth of HL-60 cells was inhibited evidently after treatment with rBTI in a dose-dependent manner, but there were minimal effects on normal human peripheral blood mononuclear cells (PBMNCs). The nuclei of HL-60 cells showed the characteristics of apoptosis, the analysis by flow cytometry indicated that the apoptosis rate of HL-60 cells was 52% after treatment with rBTI (100 microg/ml), DNA analyzed by agarose gel electrophoresis showed "ladder" pattern. It is concluded that rBTI obviously inhibits growth of HL-60 and induces its apoptosis which provides a foundation for use of recombinant common buckwheat trypsin inhibitor to cure the acute myeloid leukemia.